“…This protocol resulted in a dose–response curve for OTA in buffer with a sensitivity at 50% inhibition (IC 50 ) of 0.3 ng/mL (Fig. 2a), which is comparable to the sensitivity obtained in the indirect format [17], but more sensitive than ELISAs (IC 50 0.45–400 ng/mL) [37, 38], the used SPR biosensor immunoassay (IC 50 around 5 ng/mL) and the non-instrumental dipstick OTA assay [39] (cut-off level of 3.2 ng/mL). Fig.…”
Multi-analyte binding assays for rapid screening of food contaminants require mass spectrometric identification of compound(s) in suspect samples. An optimal combination is obtained when the same bioreagents are used in both methods; moreover, miniaturisation is important because of the high costs of bioreagents. A concept is demonstrated using superparamagnetic microbeads coated with monoclonal antibodies (Mabs) in a novel direct inhibition flow cytometric immunoassay (FCIA) plus immunoaffinity isolation prior to identification by nano-liquid chromatography–quadrupole time-of-flight-mass spectrometry (nano-LC-Q-ToF-MS). As a model system, the mycotoxin ochratoxin A (OTA) and cross-reacting mycotoxin analogues were analysed in wheat and cereal samples, after a simple extraction, using the FCIA with anti-OTA Mabs. The limit of detection for OTA was 0.15 ng/g, which is far below the lowest maximum level of 3 ng/g established by the European Union. In the immunomagnetic isolation method, a 350-times-higher amount of beads was used to trap ochratoxins from sample extracts. Following a wash step, bound ochratoxins were dissociated from the Mabs using a small volume of acidified acetonitrile/water (2/8 v/v) prior to separation plus identification with nano-LC-Q-ToF-MS. In screened suspect naturally contaminated samples, OTA and its non-chlorinated analogue ochratoxin B were successfully identified by full scan accurate mass spectrometry as a proof of concept for identification of unknown but cross-reacting emerging mycotoxins. Due to the miniaturisation and bioaffinity isolation, this concept might be applicable for the use of other and more expensive bioreagents such as transport proteins and receptors for screening and identification of known and unknown (or masked) emerging food contaminants.FigureMicrobead coated with antibody
“…This protocol resulted in a dose–response curve for OTA in buffer with a sensitivity at 50% inhibition (IC 50 ) of 0.3 ng/mL (Fig. 2a), which is comparable to the sensitivity obtained in the indirect format [17], but more sensitive than ELISAs (IC 50 0.45–400 ng/mL) [37, 38], the used SPR biosensor immunoassay (IC 50 around 5 ng/mL) and the non-instrumental dipstick OTA assay [39] (cut-off level of 3.2 ng/mL). Fig.…”
Multi-analyte binding assays for rapid screening of food contaminants require mass spectrometric identification of compound(s) in suspect samples. An optimal combination is obtained when the same bioreagents are used in both methods; moreover, miniaturisation is important because of the high costs of bioreagents. A concept is demonstrated using superparamagnetic microbeads coated with monoclonal antibodies (Mabs) in a novel direct inhibition flow cytometric immunoassay (FCIA) plus immunoaffinity isolation prior to identification by nano-liquid chromatography–quadrupole time-of-flight-mass spectrometry (nano-LC-Q-ToF-MS). As a model system, the mycotoxin ochratoxin A (OTA) and cross-reacting mycotoxin analogues were analysed in wheat and cereal samples, after a simple extraction, using the FCIA with anti-OTA Mabs. The limit of detection for OTA was 0.15 ng/g, which is far below the lowest maximum level of 3 ng/g established by the European Union. In the immunomagnetic isolation method, a 350-times-higher amount of beads was used to trap ochratoxins from sample extracts. Following a wash step, bound ochratoxins were dissociated from the Mabs using a small volume of acidified acetonitrile/water (2/8 v/v) prior to separation plus identification with nano-LC-Q-ToF-MS. In screened suspect naturally contaminated samples, OTA and its non-chlorinated analogue ochratoxin B were successfully identified by full scan accurate mass spectrometry as a proof of concept for identification of unknown but cross-reacting emerging mycotoxins. Due to the miniaturisation and bioaffinity isolation, this concept might be applicable for the use of other and more expensive bioreagents such as transport proteins and receptors for screening and identification of known and unknown (or masked) emerging food contaminants.FigureMicrobead coated with antibody
“…fat, may also cause problems. For water-soluble mycotoxins, water or buffers can be used instead [41], but in any case, additional clean-up may be needed, especially for colored samples [57]. In case of visual estimation of tests, a concentration range should be considered rather than a cut-off value which is difficult to establish due to the analog nature of both color development and human vision [41].…”
Section: Screening Methodsmentioning
confidence: 99%
“…On the other hand, the raw extract (i.e. without purification) can already yield satisfactory results in immunoassays [39, 57]. Several publications have shown that the analysis of raw extracts can be successfully accomplished by LC-MS [123, 124, 142, 152].…”
Abstract:Mycotoxins are a group of compounds produced by various fungi and excreted into the matrices on which they grow, often food intended for human consumption or animal feed. The high toxicity and carcinogenicity of these compounds and their ability to cause various pathological conditions has led to widespread screening of foods and feeds potentially polluted with them. Maximum permissible levels in different matrices have also been established for some toxins. As these are quite low, analytical methods for determination of mycotoxins have to be both sensitive and specific. In addition, an appropriate sample preparation and pre-concentration method is needed to isolate analytes from rather complicated samples. In this article, an overview of methods for analysis and sample preparation published in the last ten years is given for the most often encountered mycotoxins in different samples, mainly in food. Special emphasis is on liquid chromatography with fluorescence and mass spectrometric detection, while in the field of sample preparation various solid-phase extraction approaches are discussed. However, an overview of other analytical and sample preparation methods less often used is also given. Finally, different matrices where mycotoxins have to be determined are discussed with the emphasis on their specific characteristics important for the analysis (human food and beverages, animal feed, biological samples, environmental samples). Various issues important for accurate qualitative and quantitative analyses are critically discussed: sampling and choice of representative sample, sample preparation and possible bias associated with it, specificity of the analytical method and critical evaluation of results.
“…Enzyme-linked immunosorbent assay (ELISA) is the most common immunoassay technique used in OTA analysis due to simplicity and capability for parallel analysis of multiple samples (FujiiI, et al, 2007;Monaci and Palmisano, 2004;Visconti and De Girolamo, 2005). Even though more recently the use of biosensors allows a major portability, in situ analysis of OTA with similar selectivity and sensitivity to ELISAs and much shorter times of analysis (Goryacheva, et al, 2007;Parker and Tothill, 2009;Pohanka et al, 2007;Prieto-Simon et al, 2007). L. Bonel et al showed that big advances in mycotoxin analysis are disposable biosensors capable of measuring in situ very low concentrations of OTA with rapidly and small instrumentation.…”
Section: Figure 2 General Schematic Representation Of Biosensorsmentioning
A brief review of nanotechnology application in detection of mycotoxins and in agriculture sector was presented. Mycotoxins are secondary metabolites produced by fungi. Their toxicity is the reason for implementation of various screening methods to detect them. During the last years, the highlight was put on nanoscale materials included in biosensors, which were some of the smart devices used for determination of mycotoxins, and in agriculture sector. Over the next decade, the progress of nanotechnology will demonstrated a way to improve detection of contaminated feed and food. To achieve this purpose the innovations of nanomaterials reported every year would be applied. In the paper, some of the applications developed by nanotechnology that would contribute to the implementation of new tools for analysis of mycotoxins and agricultural products were discussed.Keywords: agriculture, biosensors, mycotoxins, nanoparticles, nanotechnology Резюме Представен е кратък преглед за приложение на нанотехнологиите за откриване на микотоксини, както и в аграрния сектор. Микотоксините са вторични метаболити, произвеждани от гъбички. Тяхната токсичност е причина за прилагане на различни методи за откриването им. През последните години акцента е бил поставен върху нано материали, вградени в биосензори, които са част от модерните устройства, използвани за откриване на микотоксини, а също и в аграрния сектор. През следващото десетилетие ще бъде доказван напредъка на нанотехнологиите да бъдат използвани като метод за детекция на замърсени фуражи и храни. За постигането на тази цел всяка година ще се следят новостите при наноматериалите. В статията са обсъдени някои от приложенията,разработени с помощта на нанотехнологиите, които биха допринесли за изграждането на нови устройства за анализ на микотоксини и селскостопански продукти.
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